© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 994-1005 doi:10.1242/dev.115352
Drosophila small heat shock protein CryAB ensures structural
integrity of developing muscles, and proper muscle and heart
Molecular chaperones, such as the small heat shock proteins (sHsps),
maintain normal cellular function by controlling protein homeostasis in
stress conditions. However, sHsps are not only activated in response
to environmental insults, but also exert developmental and tissuespecific functions that are much less known. Here, we show that during
normal development the Drosophila sHsp CryAB [L(2)efl] is
specifically expressed in larval body wall muscles and accumulates
at the level of Z-bands and around myonuclei. CryAB features a
conserved actin-binding domain and, when attenuated, leads to
clustering of myonuclei and an altered pattern of sarcomeric actin and
the Z-band-associated actin crosslinker Cheerio (filamin). Our data
suggest that CryAB and Cheerio form a complex essential for muscle
integrity: CryAB colocalizes with Cheerio and, as revealed by mass
spectrometry and co-immunoprecipitation experiments, binds to
Cheerio, and the muscle-specific attenuation of cheerio leads to
CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted
expression of CryABR120G, which carries a mutation associated with
desmin-related myopathy (DRM), results in an altered sarcomeric
actin pattern, in affected myofibrillar integrity and in Z-band breaks,
leading to reduced muscle performance and to marked cardiac
arrhythmia. Taken together, we demonstrate that CryAB ensures
myofibrillar integrity in Drosophila muscles during development and
propose that it does so by interacting with the actin crosslinker
Cheerio. The evidence that a DRM-causing mutation affects CryAB
muscle function and leads to DRM-like phenotypes in the fly reveals a
conserved stress-independent role of CryAB in maintaining muscle
cell cytoarchitecture.
KEY WORDS: Drosophila, Cheerio, CryAB, Heart, Muscle, sHsp
Most heat shock proteins operate as molecular chaperones in stress
conditions and play a central role in the maintenance of normal
cellular function by preventing the aggregation of misfolded proteins
into large deleterious complexes (Morimoto, 1998). However, some
heat shock proteins are also activated in a stress-independent way to
control the transport, folding, assembly and degradation of various
GReD - INSERM U1103, CNRS UMR6293, Clermont Université , 28, place Henri
Dunant, Clermont-Ferrand 63000, France. Department of Animal Developmental
Biology, Institute of Experimental Biology, University of Wrocław, Sienkiewicza 21,
Wrocław 50-335, Poland. Wellcome Trust/Cancer Research UK Gurdon Institute
and Department of Physiology, Development and Neuroscience, University of
Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
*Author for correspondence ([email protected])
Received 12 July 2014; Accepted 15 January 2015
proteins (Colinet et al., 2010; Lindquist and Craig, 1988; Mymrikov
et al., 2010; Sorensen et al., 2003). The cell-specific expression of
small heat shock proteins (sHsps) during development, cell
differentiation or after activation by growth factors and oncogenes
has been reported in a wide range of organisms, including Drosophila
and mammals (Michaud and Tanguay, 2003; Morimoto, 1998). For
example, the Drosophila sHsp Hsp22, in spite of its heat-induced
expression and nonspecific chaperone activity, is regulated during
normal development and plays a role in protection against apoptosis
(Colinet et al., 2010; Morrow and Tanguay, 2003).
In human, seven sHsps are expressed in cardiac and skeletal
muscles, including αB-crystallin (CRYAB), HSPB2 and HSPB3;
they are induced during the initial phase of skeletal muscle
differentiation, controlled by MYOD1, suggesting a musclespecific function in development. Also, Hspb2 and CryAB
proteins display sarcomeric localizations; they are localized on
Z-bands or I-bands of the skeletal and cardiac muscle myofibrils,
maintaining and protecting contractile cytoskeletal structures
(Mymrikov et al., 2010; Sugiyama, 2000).
CryAB displays particularly intriguing cell-specific functions,
acting as a chaperone for intermediate filament (IF) proteins such as
desmin in muscle cells and vimentin in eye lenses. IFs are known to
overlie Z-discs and M-lines, stabilizing myofibrils and protecting
them from mechanical insult (Goldfarb and Dalakas, 2009). IFs also
link membranous organelles, such as nuclei and mitochondria, and
play an important role in maintaining their correct morphology and
CryAB stabilizes IFs and prevents them from aggregating under
stress conditions, and also assists IFs during developmental
reorganizations (Nicholl and Quinlan, 1994). Importantly, mutations
in CryAB, such as replacement of arginine by glycine at conserved
position 120 (R120G), lead to altered CryAB-IF interactions
responsible for desmin-related myofibrillar myopathy (DRM)
(Dalakas et al., 2000; Goldfarb and Dalakas, 2009; Goldfarb et al.,
2004). The R120G missense mutation alters the spatial organization of
CryAB, leading to the loss of its chaperone activity and the formation
of desmin aggregates in muscle fibers (Vicart et al., 1998; Perng et al.,
2004). As a result, the desmin IF network is severely affected, leading
to altered myofibril alignment, irregular sarcomere architecture and
abnormal mitochondrial organization (Goldfarb and Dalakas, 2009).
In DRM patients this leads to progressive muscle weakness, including
the limb, neck and respiratory muscles, for which no appropriate
treatments have yet been developed.
To further characterize muscle-specific roles of CryAB and better
understand DRM-associated muscle defects we took advantage of its
evolutionary conservation to analyze the function of the Drosophila
CryAB counterpart. The fruit fly Cryab ortholog, CryAB, which is
also known as lethal (2) essential for life [l(2)efl], has been shown to
Inga Wó jtowicz1,2, Jadwiga Jabłoń ska2, Monika Zmojdzian1, Ouarda Taghli-Lamallem1, Yoan Renaud1,
Guillaume Junion1, Malgorzata Daczewska2, Sven Huelsmann3, Krzysztof Jagla1 and Teresa Jagla1,*
be involved in the modulation of insulin signaling and the regulation
of lifespan (Flatt et al., 2008) but its expression and function in
differentiated muscle have not been analyzed.
Here we show that, like its vertebrate counterpart, the Drosophila
CryAB protein displays stress-independent muscle-specific
expression. In larval muscles it accumulates around the myonuclei
and at the level of Z-bands and is required for correct nuclei
localization, myofibril integrity and muscle performance. CryAB
features a conserved actin-binding domain and is required for the
correct sarcomeric pattern of actin filaments and also of the Z-bandassociated actin crosslinker Cheerio. CryAB binds to Cheerio in
muscles, indicating that this interaction might help to maintain
myofibrillar integrity in Drosophila muscle. We also report that
muscle-targeted expression of CryAB carrying the DRM-associated
R120G mutation leads to severe alterations in the striated
actin pattern, reduced muscle performance and marked cardiac
arrhythmia. These observations point to a conserved stressindependent role of CryAB in the maintenance of muscle cell
cytoarchitecture. Thus, our findings provide evidence that the
Drosophila sHsp CryAB shares not only sequence but also
functional similarities with its human counterpart, suggesting that
Drosophila can serve as a model system for dissecting molecular
determinants of myofibrillar myopathies.
Drosophila CryAB is a conserved sHsp with stressindependent expression in larval body wall muscles
The Drosophila sHsp genes are clustered within the 67B region on
the left arm of the third chromosome (Corces et al., 1980), except for
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
CryAB [l(2)efl], which is located on the right arm of the second
chromosome (Kurzik-Dumke and Lohmann, 1995). Drosophila
CryAB shows significant homology to human CRYAB (Fig. 1A)
and to other sHsp members, in particular the Hspb1 and Hspb8
subfamilies (Fink et al., 2009; McWilliam et al., 2013) (Fig. 1C).
CryAB encodes a protein of ∼22 kDa (Fig. 1B) that has a highly
conserved α-crystallin domain located in the C-terminal portion of
the protein (Fig. 1A), similar to other sHsps (Ingolia and Craig,
1982). Within this domain, CryAB features several conserved
motifs, including an actin-binding domain, a capping box and the
residues R120 and D109 involved in desminopathies (Fig. 1A).
In addition to coordinately induced expression in response to a
heat stress, several members of the sHsp family show a specific
pattern of expression in diverse tissues and cells (Marin and
Tanguay, 1996; Michaud and Tanguay, 2003). To determine
whether CryAB also displays stress-independent expression during
development we first analyzed its expression pattern in third instar
larvae. Using a polyclonal antibody raised against the whole CryAB
protein, we found that CryAB is abundantly expressed in the larval
body wall muscles (Fig. 2). Within an individual larval muscle,
CryAB accumulated in the perinuclear area (Fig. 2A) and displayed
a striated sarcomeric pattern at the level of Z-lines and M-lines
(Fig. 2A-D). We confirmed this observation by the immunogold
detection of CryAB on the ultrathin sections visualized in electron
microscopy (EM) (supplementary material Fig. S1). When
analyzing CryAB distribution on longitudinal optical sections
through the muscle fibers, we also found that it accumulated around
the nuclei and enveloped the external surface of myonuclei
(Fig. 2E). We observed a granular pattern of CryAB both around
Fig. 1. Drosophila CryAB is a highly conserved
member of the CryAB sHsp family that is
phylogenetically related to the Hspb1 and
Hspb8 sHsp families. (A) Protein sequence
alignment of Drosophila and human CryAB
proteins. Conserved amino acid residues are in red
and conserved functional domains are highlighted
by boxes or underlining. (B) Western blot of third
instar larvae protein extract showing the 22 kDa
band of the expected size of CryAB protein.
(C) Phylogenetic tree of the related CryAB, Hspb1
and Hspb8 families.
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
the nuclei (Fig. 2E) and at the Z-discs (Fig. 2B,D), indicating that it
might be part of large protein complexes. 3D reconstruction of
confocal scans (Fig. 2F) revealed that the Z-line-associated CryAB
is mainly localized at the external level of Z-discs. This
developmental expression pattern of Drosophila CryAB is
reminiscent of that of its human ortholog (Dubin et al., 1990).
CryAB is required for Z-band patterning and muscle integrity
To assess the role of CryAB in larval muscles we applied
RNAi-mediated muscle-specific gene attenuation (Fig. 3I) using
two different UAS-CryAB-RNAi lines (VDRC 40532 and 107305)
crossed to the Mef2-Gal4 driver (Fig. 3; supplementary material
Fig. S2). We observed that most Mef2>CryAB-RNAi larvae (from
both RNAi lines examined) exhibited defects in sarcomeric
organization, characterized by an irregular, fuzzy pattern of
phalloidin staining in the large muscle segments (Fig. 3C,H;
supplementary material Fig. S2). This aberrant organization of
sarcomeric actin is consistent with the fact that CryAB has an actinbinding domain (ABD) (see Fig. 1), which appears to play an
important role in actin filament organization assuming that muscletargeted expression of CryAB carrying a mutated ABD leads
to RNAi-like phenotypes (supplementary material Fig. S3).
Moreover, CryAB attenuation also leads to muscle splitting
(Fig. 3B; supplementary material Fig. S4A), which in some cases
is associated with altered muscle attachments (Fig. 3B) or even with
Fig. 3. Muscle-specific attenuation of CryAB leads to morphological
muscle defects and disrupted sarcomeric Z-band pattern. (A) Lateral view
of wild-type third instar larval muscles stained with phalloidin. (B,C) Effects of
muscle-targeted RNAi attenuation of CryAB. Arrows in B indicate splitting of
myofibers. Arrow in C denotes fuzzy Z-band pattern; compare with wild-type
muscles (asterisks in A). (D) A portion of wild-type VL3 muscle showing a
regular pattern of Msp300 co-stained with phalloidin. (E) Representative view
of a portion of VL3 and VL4 muscles from Mef2>CryAB-RNAi animals. Arrows
point to muscle areas with a fuzzy actin pattern in which Msp300 labeling is
reduced or missing. (F) EM view of wild-type VL4 sarcomeres. (G) Similar view
from Mef2>CryAB-RNAi larvae showing interrupted Z-bands. Yellow
arrowheads point to Z-bands, which are also traced by the red line. Asterisks
indicate electron-dense accumulations of glycogen. Double-ended arrows
indicate the extension of sarcomeres. (H) Statistical representation of muscle
defects observed in Mef2>CryAB-RNAi larvae. (I) RT-PCR analysis of CryAB
attenuation in Mef2>CryAB-RNAi versus wild-type (Wt) larvae. Gapdh
provides a loading control. Scale bars: 20 µm in A-E; 2 µm in F,G.
the loss of affected muscle (supplementary material Fig. S4B). The
observed muscle defects are not due to apoptotic events, as we do not
detect Caspase 3 activation associated with muscles in the CryABRNAi context (supplementary material Fig. S2I). Altogether, these
Fig. 2. CryAB displays Z-band-associated and perinuclear expression in
larval muscles. (A,C) CryAB expression at the superficial level of external
myofibrils. Note the accumulation of CryAB at the level of Z-bands (arrowhead)
and around the nuclei (arrow). (B,D) CryAB expression within the myofiber.
CryAB protein is detected at the Z-bands (white arrowhead) but also in M-lines
(yellow arrowhead). (E) A portion of larval segment border muscle at the level
of the nuclei. Note high CryAB accumulation in a perinuclear area (arrow) and
at the external surface of the nucleus. (F) Imaris-rendered 3D reconstruction of
confocal scans through ventral VL3 larval muscle stained for CryAB and with
phalloidin (F-actin). Note the external localization of CryAB on the top of
phalloidin-stained Z-bands (arrowhead). Asterisks indicate nuclei. Scale bars:
10 µm.
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
findings demonstrate a role of CryAB in the maintenance of muscle
fiber integrity.
To better characterize the influence of CryAB attenuation on the
sarcomeric pattern we used Msp300 antibody to reveal the Z-bands
(Fig. 3D,E). We found that, in the muscle segments displaying fuzzy
phalloidin staining, the Z-band-associated signal of Msp300 was weak
or lacking (Fig. 3E). We also noted that Msp300 was irregularly
distributed around the myonuclei (Fig. 3D,E), suggesting that it might
potentially interact with CryAB. We tested this possibility by
co-immunoprecipitation (co-IP) (supplementary material Fig. S5)
and found that there is no physical interaction between these two
Z-disk-associated proteins. Gaps in Z-bands were also detected when
examining EM sections of CryAB attenuated muscles (Fig. 3F,G),
supporting the view that CryAB acts to stabilize Z-band-associated
sarcomeric components.
Assuming that each of the larval muscles displays a specific
number of sarcomeres (Bate, 1990), we also tested whether muscletargeted CryAB knockdown impacted on sarcomere number. We
focused on three distinct muscles (VL3, VL4 and SBM) and found
that, in all cases, the number of sarcomeres was significantly reduced
(supplementary material Fig. S4A), whereas, except for the fuzzy
pattern areas, the size of sarcomeres was unaffected (supplementary
material Fig. S4B). Thus, CryAB also appears to play a role in larval
muscle growth, which occurs by the addition of sarcomere units.
Next we investigated whether the cellular organization and the
performance of muscles are affected by the reduction of CryAB
function. Previous studies demonstrated that the localization of
nuclei is crucial for proper muscle performance (Metzger et al.,
2012) and that mitochondria are highly organized in muscles
(Picard et al., 2012). The accumulation of CryAB around the nuclei
(Fig. 2) prompted us to test whether it was involved in the correct
distribution of myonuclei within the muscle fibers. We found that in
the CryAB-RNAi context they were irregularly distributed along the
muscle fibers (Fig. 4A,B). Nuclei are also misarranged in muscles
overexpressing CryAB with a mutated ABD (supplementary
material Fig. S3), providing evidence that the actin binding
function of CryAB is essential for nuclei localization.
Cheerio, which makes a link between perinuclear actin and
cytoplasmic actin cables, is involved in nuclei positioning in
Drosophila nurse cells (Huelsmann et al., 2013). It is also well
known that in vertebrate muscles the Cheerio ortholog Filamin C is
associated with Z-bands and when mutated leads to myofibrilar
myopathies (Goldfarb and Dalakas, 2009). We tested whether
Cheerio is expressed in larval muscles and whether CryAB
influences its localization. Cheerio was detected aligned with
Z-bands (Fig. 4C), but its pattern was more expansive than the sharp
Z-line revealed by phalloidin. As in nurse cells, it also accumulated
in the perinuclear area. However, in muscles attenuated for CryAB
the sarcomeric localization of Cheerio was severely affected, with
pronounced accumulation around the mislocalized nuclei (Fig. 4D),
strongly suggesting that CryAB is required for Cheerio localization.
In addition, the mitochondrial signal appeared significantly reduced
in CryAB-RNAi muscles compared with the wild type (Fig. 4E,F),
indicating impaired respiratory functions. This possibility was
supported by EM analysis revealing mitochondrial swelling, broken
cristae and glycogen deposits (Fig 4G,H).
To test whether the described morphological alterations affected
the locomotive abilities of larvae, we carried out three behavioral
tests. We measured the speed of crawling and found that
Fig. 4. CryAB is required for muscle integrity and performance.
(A) Distribution of nuclei in wild-type VL3 and VL4 muscles. VL3 muscle
diameter is indicated by the yellow line. A slightly reduced number of nuclei is
observed in muscles with attenuated CryAB, which could be due to
destabilization of myofibrillar architecture and subsequent loss of some of
mislocalized nuclei. (B) Mef2>CryAB-RNAi VL3 muscles displayed reduced
diameter (yellow line) and mislocalized nuclei (arrows). (C) Wild-type pattern of
Z-band-associated Cheerio. Asterisks indicate nuclei. (D) Disrupted Cheerio
pattern in Mef2>CryAB-RNAi context. (E) Wild-type pattern of mitochondria
revealed by MitoTracker Red. (F) Attenuation of CryAB leads to a reduced
MitoTracker signal. (G) EM view of wild-type third instar muscle with functional
mitochondria. (H) EM view of a Mef2>CryAB-RNAi muscle with
morphologically abnormal mitochondria. Arrowheads indicate mitochondria
and asterisks indicate the Z-band. (I-L) Muscle performance tests showing
reduced motility and affected contractility of Mef2>CryAB-RNAi larvae.
Error bars indicate s.e.m.; n=30 flies per genotype *P<0.05 (one-way ANOVA).
Scale bars: 20 µm in A,B; 10 µm in C,D; 5 µm in E,F; 1 µm in G,H.
CryAB-RNAi larvae needed ∼20% more time to travel the
appointed distance (Fig. 4I). We also measured the time needed
for each larva to right itself from the dorsal to ventral position. In this
test, CryAB-attenuated individuals took twice as long (Fig. 4J).
Finally, the number of peristaltic movements of crawling larvae was
counted: during a 30 s period, CryAB-RNAi specimens executed at
CryAB is required for correct myonuclei localization,
mitochondrial organization and muscle performance
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
least 20% fewer peristaltic movements than controls (Fig. 4L). To
confirm the contraction disturbance, we also compared the length of
muscle fibers relaxed by EDTA with that of contracted muscle
fibers. This showed that the analyzed muscles from CryAB-RNAi
individuals had a significantly lower contractility index (Fig. 4K).
Thus, these data show that CryAB is required for proper muscle
contraction and efficient motility. Finally, we found that these
functions of CryAB in muscles also influence the survival of flies
(supplementary material Fig. S3C).
The R120G mutation of DRM patients leads to a dominantnegative CryAB protein and affects sarcomeric pattern and
muscle performance
DRM is caused by the missense R120G mutation within the
α-crystallin domain of CRYAB. It is due to intracellular
aggregations of desmin and Vimentin IFs, which abnormally
interact with the mutated CRYAB (Simon et al., 2007; Zobel et al.,
2003). Comparison of amino acid sequences of human and
Drosophila CryAB proteins showed conservation of arginine at
position 120 (Fig. 1A) and prompted us to test whether the R120G
mutation in Drosophila CryAB could mimic muscle defects
observed in DRM patients. We generated transgenic lines carrying
either the wild-type or mutated form of CryAB fused to GFP
(Fig. 5A-D″).
Compared with endogenous CryAB (Fig. 2), both CryAB-GFP and
CryABR120G-GFP displayed irregular accumulations along the muscle
fibers in addition to their localization in sarcomeres (Fig. 5A′-D′).
We therefore tested whether these accumulations affected
sarcomeric organization. In the area where the wild-type CryABGFP accumulated, the sarcomeric pattern marked with phalloidin
Fig. 5. Defective structural integrity and muscle
performance induced by muscle-targeted
expression of CryABR120G. (A-A″) Z-band pattern
in the muscle area with accumulation of wild-type
CryAB-GFP. Note the Z-line shift (arrowheads,
magnified in inset) close to the spot of CryAB-GFP
accumulation. (B-B″) Irregular phalloidin staining in
muscle expressing CryABR120G-GFP. Note that
muscle portions with high levels of CryABR120GGFP display a weak phalloidin signal with an
increased distance between Z-bands
(arrowheads), whereas the areas devoid of
CryABR120G-GFP show a condensed sarcomeric
pattern (arrows) with high phalloidin signal.
(C-C″) Muscle portion with targeted expression of
CryABR120G-GFP showing aggregate-like pattern of
GFP (arrows). (D-D″) A detailed view of sarcomeric
localization of CryABR120G-GFP expressed under
the Mef2-Gal4 driver. Note that CryABR120G is
detected in Z-lines and also in two bands on each
side of the Z-disk. Arrowhead indicates Z-line
shift that coincides with the accumulation of
CryABR120G-GFP. (E-H) Muscle performance tests
showing reduced motility and affected contractility
of Mef2>CryABR120G-GFP larvae. Error bars
indicate s.e.m.; n=28 flies per genotype; *P<0.05,
**P<0.005, ***P<0.0005 (one-way ANOVA). Scale
bars: 30 µm in A,B; 20 µm in C; 10 µm in D.
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
Fig. 6. Cardiac-specific attenuation of CryAB and
expression of CryABR120G affect heart performance.
(A) Dorsal view of dissected adult heart from a 1-week-old fly
stained for CryAB and with phalloidin (left panels show an
overview). CryAB is expressed in longitudinal fibers
associated with the heart (middle panels) and the transverse
myofibrils of the cardiac muscle (right panels). (B-D) Heart
period (B), arrhythmia index (C) and systolic diameter (D) of
1-week-old flies of the indicated genotypes. Note that
knockdown of CryAB shows significant shortening in the heart
periods (B). Upon expression of CryABR120G, the arrhythmia
index is substantially increased (C) and systolic diameter is
enlarged (D). Error bars indicate s.e.m.; N=20 flies per
genotype and age. *P<0.05, **P<0.005, ***P<0.0005 (oneway ANOVA). (E) Representative M-mode traces (5 s)
illustrating movements of heart tube walls ( y-axis) over time
(x-axis). Movement traces of cardiac tubes expressing CryABRNAi illustrate the increased heart rate and the rhythmic
beating patterns. M-modes from hearts expressing
CryABR120G reveal arrhythmic beating patterns. Diastolic (red)
and systolic (black) intervals are indicated in each M-mode
trace. Scale bars: 200 µm in A, left; 25 µm in A, middle and
these tests (Fig. 5E-G), Mef2>CryABR120G larvae showed markedly
reduced muscle performance than those expressing wild-type
CryAB in muscles. We also found that muscle-targeted expression
of CryABR120G affected muscle contractility (Fig. 5H). The fact
that several phenotypes of CryABR120G overexpression are similar
to those observed after knocking down CryAB suggests that
CryABR120G acts as a dominant negative.
Cardiac-specific attenuation of CryAB and expression of
CryABR120G impair heart performance
A recent study demonstrated that when the mutated human CRYAB
is expressed in adult Drosophila heart it mimics DRM-related
cardiac defects such as arrhythmia and an increase in systolic heart
diameter (Xie et al., 2013). However, it remained unclear whether
these defects were due to reduced functions of the endogenous
Drosophila CryAB gene. We therefore tested whether CryAB is
expressed in the adult Drosophila heart and if it is required for
normal heart function.
We found that CryAB protein is present in a striated pattern in
both the transverse myofibrils of the myocardium and in the layer of
the longitudinal muscle fibers that is associated with the heart
(Fig. 6A). This CryAB expression plays an important role in the
adult heart because the heart-specific attenuation of CryAB resulted
in an accelerated cardiac rhythm (Fig. 6B,E). The video-captured
M-modes of hearts revealed a reduction in the heart period
following RNAi-mediated CryAB knockdown, as compared with
appeared normal, although the intensity of actin bands was
attenuated (Fig. 5A-A″, asterisk). Occasionally, we observed
misalignments of myofibrils (Fig. 5A, arrowheads), suggesting
that the overexpression of CryAB leads to a local destabilization of
myofibrillar organization. In contrast to these mild phenotypes, the
accumulations of CryABR120G resulted in severely altered sarcomeric
patterns (Fig. 5B-D″), with virtually all muscles affected. We
observed a striking complementary distribution of CryABR120G with
respect to sarcomeric actin along the muscles. Large segments of
muscle fibers with high levels of CryABR120G displayed reduced
phalloidin staining with abnormally interspaced and irregular
Z-bands (Fig. 5B, arrowheads), whereas the neighboring
CryABR120G-free regions were characterized by a high-intensity
phalloidin signal probably resulting from the disorganization and
local compaction of sarcomeres (Fig. 5B, arrows). In addition to
localizing to the Z-bands, CryABR120G accumulated in either a fuzzy
pattern (Fig. 5B′,B″) or a punctate pattern suggesting the formation of
aggregates (Fig. 5C′,C″). We also observed small patches of
accumulated CryABR120G (Fig. 5D-D″) in which the sarcomeric
pattern was not particularly affected. However, we noted that within
each sarcomere CryABR120G was not only present in the Z-bands
but also in two additional stripes between the Z-bands (Fig. 5D″),
and that myofibrils were misaligned (Fig. 5D, arrowheads).
These alterations prompted us to test whether the mutated form of
CryAB also affected larval muscle function. We tested muscle
performance using the righting, motility and journey tests. In all
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
Fig. 7. CryAB interacts with Cheerio. (A) Schematic
representation of the cher-240 and cher-90 isoforms. Asterisks
indicate the location of peptides identified by mass spectrometry.
The bar beneath indicate the region recognized by anti-Cheerio
antibody. (B) (Left) Western blot showing the different Cheerio
isoforms detected in protein extract from third instar larvae. (Right)
Western blot from IP with anti-CryAB antibody on protein extract
from third instar Mef2>cher-90-GFP larvae. Anti-GFP antibody was
used to reveal cher-90-GFP. (C) cher-240 with Venus insertion
colocalizes with CryAB in Z-bands. (D) Accumulations of cher-90
overexpressed in larval muscles coincide with increased levels of
CryAB (arrows), confirming interactions between the two proteins
in vivo. (E) Muscle-targeted attenuation of cheerio leads to the
fuzzy sarcomeric actin pattern (asterisks) and to the loss of Z-bandassociated localization of CryAB. Note the CryAB aggregates
(arrows) that coincide with the fuzzy actin pattern (asterisk).
The boxed region is shown at higher magnification to the right.
Scale bars: 10 µm in C; 20 µm in D,E.
analysis reveals a novel role of CryAB in the regulation of heartbeat
and demonstrates that introducing the R120G mutation into
Drosophila CryAB leads to pathological cardiac arrhythmia and
mimics cardiac defects observed in DRM patients.
CryAB interacts with Cheerio in larval muscles
The altered Cheerio pattern in larval muscles with attenuated CryAB
prompted us to test whether these two proteins physically interact.
We first performed a co-IP experiment with anti-CryAB antibody
followed by proteomics analyses. Peptide sequencing of coimmunoprecipitated proteins ranging from 30 to 170 kDa revealed
that not only actin but also Tropomyosin 1 and 2 and a few other
sarcomeric components, including α-Actinin, Myosin heavy chain
and Paramyosin, were strongly represented in CryAB-containing
protein complexes (supplementary material Table S1). Importantly,
mass spectrometry data also revealed nine matches in Cheerio,
corresponding to six different peptides all located in the C-terminal
control flies (Fig. 6E). The heart period was strongly reduced in
young flies (Fig. 6B,E) and continued to be significantly shortened
in aged Hand>CryAB-RNAi animals (data not shown). The
shortened heart periods in CryAB-attenuated flies were mainly
due to decreased diastolic and systolic intervals (Fig. 6E;
supplementary material Fig. S6). Conversely, the heart periods of
flies overexpressing the wild-type or R120G form of CryAB were
unaffected or only slightly (non-significantly) decreased (Fig. 6B).
However, heart-specific expression of CryABR120G led to a
significant increase of arrhythmia in 1-week-old flies (Fig. 6C), a
phenotype similar to that observed in transgenic Drosophila lines
expressing the mutated form of human CRYAB in the heart (Xie
et al., 2013). Additionally, altering the expression of CryAB or
introducing CryABR120G in the heart resulted in enlarged heart
diameter (Fig. 6D; supplementary material Fig. S6) and significantly
reduced the fractional shortening in CryABR120G flies, impairing
systolic function (supplementary material Fig. S6). Thus, this
region of the protein (Fig. 7A). cheerio (cher) is the Drosophila
homolog of human filamin (Sokol and Cooley, 1999) and encodes
several isoforms ranging from 90 to ∼260 kDa, all of which are
present in third instar larvae (Fig. 7B). The cher-90 isoform is
devoid of an ABD and the Rod1 repeat region, and is made up of the
C-terminal part of cher-240 (Fig. 7A). Since all the identified
peptide sequences matched this common C-terminal part, each
Cheerio isoform can potentially interact with CryAB.
To confirm the proteomics data, we performed co-IP experiments
with anti-CryAB antibody using protein extracts from the dissected
third instar larvae expressing GFP-tagged cher-90 in muscles. We
found that immunoprecipitated CryAB protein complexes did
indeed contain Cheerio (Fig. 7B). We also tested whether CryAB
colocalized with cher-240 in sarcomeres using the cherCPTI0847 line,
which carries a Venus insertion that does not affect cheerio function
(Huelsmann et al., 2013). We observed that Venus-tagged Cheerio
colocalized at the level of Z-bands with CryAB (Fig. 7C).
Moreover, when we overexpressed cher-90-GFP in muscles, it
colocalized with CryAB (Fig. 7D), supporting the view that CryAB
interacts with Cheerio. Importantly, Cheerio not only interacts with
CryAB but also appears to stabilize sarcomeric cytoarchitecture in a
manner similar to that of CryAB: knockdown of cheerio leads to the
fuzzy actin pattern associated with the aggregate-like accumulations
of CryAB (Fig. 7E).
To test functional relationships between CryAB and Cheerio
we generated a double UAS-cherRNAi;UAS-CryABRNAi line that
allows attenuation of both genes simultaneously and a rescue line
by combining the UAS-cherRNAi line with UAS-CryAB. The
sarcomeric actin pattern in cheerio CryAB double-knockdown
muscles is severely affected, with areas of weak phalloidin
staining complemented by an abnormal Z-band-associated
accumulation of Kettin (Sallimus – FlyBase) (supplementary
material Fig. S7A-A″), a phenotype that has not been observed in
a simple CryAB-RNAi context (Fig. 4B). Targeted overexpression
of CryAB in muscles with attenuated cheerio partially rescues
proper sarcomeric actin organization and fully restores the normal
Kettin pattern (supplementary material Fig. S7B-B″). Thus,
CryAB and Cheerio not only bind to each other but also
interact genetically to stabilize sarcomeric and, potentially,
perinuclear actin. Altogether, these experiments show that
CryAB-Cheerio interactions are crucial for the maintenance of
the contractile apparatus in muscles and suggest that loss of this
interaction contributes to the muscle defects and affected muscle
performance observed in CryAB-RNAi larvae.
It has been demonstrated that unfolded filamin interacts with
Drosophila/mammalian co-chaperone Starvin/BAG3, which plays
an important role in the maintenance of Z-disk components (Arndt
et al., 2010). We tested whether knocking down starvin in muscles
influences CryAB sarcomeric localization. The attenuation of
starvin led to Z-band breaks and an irregular sarcomeric actin
pattern associated with clustering of myonuclei (supplementary
material Fig. S8). However, the CryAB sarcomeric pattern and
perinuclear localization appeared unaffected (supplementary
material Fig. S8), supporting a view that CryAB-Cheerio and
Starvin-Cheerio interact independently. Since the CRYAB-filamin
C interaction has not as yet been tested in vertebrate skeletal
muscles, whether this interaction is a conserved feature of CryAB
proteins remains to be elucidated.
The physiological response to stressful conditions in muscle tissue
involves high-molecular-mass Hsps such as Hsp70 (McArdle et al.,
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
2004), together with a set of sHsps (Koh and Escobedo, 2004;
Haslbeck et al., 2005). However, besides this classical role, in recent
years stress-independent and tissue-specific expression and function
have been reported for several members of the Hsp gene family
(Michaud and Tanguay, 2003; Rupik et al., 2011).
Here, we provide evidence that in Drosophila, the sHsp
subfamily member CryAB [which is also known as l(2)efl
(Kurzik-Dumke and Lohmann, 1995)], which encodes the fruit
fly ortholog of mammalian CRYAB, is specifically expressed in
developing larval somatic muscles and is required for myofibril
integrity and muscle performance.
The tissue-specific expression and subcellular localization
of CryAB are reminiscent of its vertebrate counterpart
In the developing mammalian heart and skeletal muscles, CRYAB
and six other sHsps (HSPB1, HSPB2, HSPB3, HSPB6, HSPB7 and
HSPB8) are expressed at a relatively high levels (Davidson et al.,
2002). It is not known whether all these genes diverged from a
common ancestor with developmental functions, but our phylogenetic
analysis indicates that two sHsps involved in muscle development,
Hspb8 and CryAB, do have a common ancestor. We also showed that,
like its vertebrate counterpart (Doran et al., 2007), the fruit fly CryAB
displays muscle- and heart-specific expression and accumulates at the
level of the Z-bands and around the nuclei. Interestingly, Drosophila
CryAB is also localized in between the Z-bands and muscle cell
membranes and displays a punctate expression pattern that suggests
that it might be a component of Z-band-associated protein complexes.
It is well known that vertebrate CRYAB is also expressed at the level of
Z-bands and, by acting as a chaperone of the type III IF protein desmin,
helps to maintain cytoskeletal integrity in skeletal and cardiac muscle
cells (Golenhofen et al., 1999). Desmin IFs are mainly associated with
Z-bands and interact with other sarcomeric proteins to form a
continuous cytoskeletal network that maintains the spatial
organization of contractile apparatus (Goldfarb and Dalakas, 2009).
However, neither desmin nor vimentin (another type III IF protein)
orthologs exist in Drosophila (Sparrow and Schöck, 2009),
suggesting that other cytoskeletal components of muscle cells
replace them functionally to ensure myofibrillar integrity. The
subcellular localization of Drosophila CryAB strongly suggests that
it interacts with sarcomeric components and, in particular, with
Z-band-associated proteins, thereby helping to maintain myofibrillar
Muscle-specific knockdown of CryAB reveals its stressindependent role in sarcomeric organization, muscle
performance and heart rhythm
Point mutations in human sHsps lead to several aggregation diseases
(Clark and Muchowski, 2000). For example, mutations in αAcrystallin (CRYAA) lead to cataract (Litt et al., 1998), missense
mutations in HSP27 (HSPB1) are associated with Charcot-MarieTooth disease (Evgrafov et al., 2004), and point mutations in
CRYAB cause DRM (Vicart et al., 1998; Sacconi et al., 2012). Yet,
the stress-independent functions of individual members of the sHsp
gene family have not been systematically assessed in animal models
because of functional redundancies and compensation effects. In the
case of Cryab, mice knocked out for this gene also lack the adjacent
Hspb2 gene and display no obvious developmental defects (Brady
et al., 2001). However, as they become older, Cryab Hspb2
homozygous knockout mice show postural defects and other health
problems that appear to stem from progressive myopathy (Brady
et al., 2001). For Drosophila CryAB, the impact of its loss of
function and its lethality have not yet been confirmed.
Here, we analyzed the effects of muscle- and heart-targeted
knockdown of Drosophila CryAB to better evaluate its stressindependent functions. We focused on larval musculature and on the
adult heart, and found that attenuation of CryAB led to a number of
morphological and ultrastructural defects in body wall muscles,
impaired muscle performance and accelerated heartbeat. Importantly,
muscle alterations lead to local Z-band misalignment, which could be
responsible for the observed muscle fiber splitting. Affected
myofibrillar integrity results in a markedly altered contractility
index in Mef2>CryAB-RNAi larvae, which also display lower motility
compared with age-matched wild-type individuals. The sarcomeric
alterations are also associated with the clustering/mislocalization of
nuclei in muscle fibers and with abnormal swelling of mitochondria,
together revealing the important developmental role of CryAB in
Drosophila in the maintenance of correct muscle function and
structural integrity. Some of the phenotypes resulting from the
muscle-specific attenuation of Drosophila CryAB, including
disruption of myofibrillar organization and muscle weakness, are
reminiscent of those observed in myofibrillar myopathies in human
(Goldfarb and Dalakas, 2009).
Body wall and cardiac muscle alterations in larvae
expressing CryAB with the R120G mutation
In human pathological cases, when the IF network does not
assemble correctly and creates aggregates, CRYAB can reorganize
the malfolded IF proteins into a normal filamentous network
(Koyama and Goldman, 1999). Some mutations in CRYAB, such as
R120G (Vicart et al., 1998) or D109H, are claimed to affect
dimerization of the protein (Sacconi et al., 2012) leading to loss of
its chaperone activity and ultimately to pathological muscle defects.
Interestingly, protein sequence alignment revealed that both these
residues (R120 and D109) are conserved in Drosophila CryAB.
When we generated a Drosophila strain carrying a mutation in one
of these residues, CryABR120G, we found that in larval muscle cells
with accumulations of CryABR120G the sarcomeric architecture and,
in particular, the actin filament pattern were severely affected. We
also observed a misalignment of myofibrils and the appearance of
protein aggregates containing CryABR120G, symptoms also seen
in DRM patients (Goldfarb and Dalakas, 2009). The affected
myofibrillar integrity and irregular sarcomeric pattern correlated
with reduced muscle performance as measured by motility assays in
Mef2>CryABR120G third instar larvae. Taken together, these
observations suggest that the R120G mutation in Drosophila
CryAB has an impact on larva body wall muscles similar to that
which the analogous mutation in CRYAB has on human skeletal
muscles leading to disruption of myofibrillar integrity and muscle
weakness. In DRM patients carrying the CRYABR120G mutation,
muscle defects appear in mid-age adults, whereas in our Drosophila
model, following forced expression of CryABR120G in muscles, they
are apparent in third instar larva. At this late stage of larval
development muscles are fully functional and differentiated, like
those in adult humans.
Another important phenotype observed in patients carrying the
R120G mutation in CRYAB is increased systolic heart diameter,
dilated cardiomyopathy and arrhythmia. Here again, targeted
cardiac expression of CryABR120G resulted in arrhythmic heart
beating in the adult fly, reminiscent of that observed in the DRM
mouse model and in DRM patients (Wang et al., 2001).
Thus, expressing the R120G-mutated form of CryAB in body
wall or in cardiac muscles in Drosophila leads to pathological
defects similar to those observed in patients harboring the
CRYABR120G mutation. This suggests that this mutation, which
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
potentially impairs CryAB dimerization (Sacconi et al., 2012),
might have a more general impact on the chaperone function of
CryAB and not only on interactions with desmin IFs.
Cheerio and other potential CryAB-interacting proteins in
Drosophila muscles
As reported above, muscle-specific expression of Drosophila
CryABR120G leads to DRM-like phenotypes with Z-band
disruption and adversely affected muscle performance. However,
as revealed by sequencing of the Drosophila genome (Adams et al.,
2000), no gene orthologous to desmin has been identified in the fruit
fly, suggesting that CryAB interacts with other sarcomeric
components to stabilize the contractile apparatus. To identify
potential CryAB-interacting proteins we performed co-IP
experiments using dissected third instar larvae. Highly enriched
sarcomeric proteins bound by CryAB include actins, Tropomyosin
1 and 2, α-Actinin, Paramyosin, Myosin heavy chain and Cheerio.
Drosophila CryAB accumulates in Z-bands and to a lesser extent
in M-lines, and thus can potentially interact with all these
sarcomeric components. Identifying actin in the IP material is
consistent with the fact that CryAB has an ABD and that the actin
pattern is affected in muscle fibers with attenuated CryAB as it is in
muscles expressing CryAB carrying a mutation in the ABD.
However, the finding that CryAB can potentially interact with
Z-band-associated Cheerio was of particular interest, as in humans
mutations in filamin C lead to myofibrillar myopathy with
dissolution of myofibrils similar to that observed in DRM (Kley
et al., 2007; Luan et al., 2010). Filamins are actin-crosslinking
proteins consisting of an N-terminal ABD followed by 24
immunoglobulin-like repeats (Stossel et al., 2001) and are
involved in a number of cellular processes including cell-matrix
adhesion, mechanoprotection and actin remodeling (Feng
and Walsh, 2004). In vertebrates, filamin C is muscle specific
and localizes at myotendinous junctions and also at Z-bands and
costameres (Ohashi et al., 2005; van der Ven et al., 2000a,b). It has
been shown that filamin C interacts with two major protein
complexes at the sarcolemma, namely the dystrophin-associated
glycoprotein complex (DGC) (Thompson et al., 2000) and the
integrin complex (Gontier et al., 2005; Loo et al., 1998), both of
which are known to have important roles in affording mechanical
integrity to striated muscle. However, through its C-terminal
region, filamin C also binds the Z-band proteins myotilin (van der
Ven et al., 2000b) and myopodin (synaptopodin 2) (Linnemann
et al., 2010), suggesting that it ensures a link between Z-bands and
sarcolemmal DGC and integrin complexes, thus maintaining the
mechanical integrity of muscle cells. This possibility is supported
by the detachment of myofibrils from sarcolemma and intercalated
Z-bands in muscles of zacro (filamin C) medaka mutants (Fujita
et al., 2012). In Drosophila, overall filamin protein structure its
actin-crosslinking function are conserved in Cheerio. It plays a key
role in ring canal formation during oogenesis (Sokol and Cooley,
1999) and is involved in the positioning of nuclei in ovarian nurse
cells (Huelsmann et al., 2013), but the role of Cheerio in muscles
had remained elusive.
Here, we show that in third instar larval muscles Cheerio protein
accumulates between myofibrils and the muscle cell membrane at
the level of Z-bands and around the nuclei, a subcellular localization
reminiscent of that of filamin C. We also found that the sarcomeric
Cheerio pattern was disrupted in larvae with attenuated CryAB,
suggesting that the identified interactions between CryAB and
Cheerio might help to maintain myofibrillar integrity. Thus, we
hypothesize that CryAB exerts a chaperone function on Cheerio in
Drosophila muscles, which are devoid of desmin IFs, and that this
interaction helps to stabilize the contractile apparatus.
Recently, it was reported that the conserved BAG3/Starvin complex
detects mechanical unfolding of filamins caused by cell stretching
(Arndt et al., 2010; Ulbricht et al., 2013). This complex binds unfolded
filamins and targets them to autophagosomes for degradation. It is
unclear whether Drosophila CryAB is part of the same complex or of
another sHsp complex that regulates filamin and muscle cell function.
The fact that Drosophila CryAB can interact with cher-90, the Cheerio
isoform that does not contain an ABD and thus might not be stretched
and mechanically opened up, suggests that the CryAB complex might
bind the folded C-terminus of Cheerio and thus not be part of the
BAG3/Starvin complex. Future experiments are required to determine
whether the CryAB and BAG3/Starvin complexes bound to filamins
have overlapping or specific roles during the development and function
of skeletal and cardiac muscle cells.
These findings raise the question as to the extent to which Cheerio
is able to replace the IF network in Drosophila muscles. Human
CRYAB is known to interact with desmin (Goldfarb and Dalakas,
2009), with sarcomeric actin (Bennardini et al., 1992) and with titin
(Golenhofen et al., 2002); however, whether the identified
interaction between Drosophila CryAB and Cheerio is conserved
in human muscles and, if so, whether it helps to stabilize
myofibrillar architecture remains to be elucidated.
Development (2015) 142, 994-1005 doi:10.1242/dev.115352
Methods. The heart physiology assay was performed as described
previously (Fink et al. 2009).
Immunofluorescence staining of larval muscles and adult fly
Third instar larvae were dissected as described previously (Budnik et al.,
2006) in ice-cold Ca2+-free saline buffer containing 128 mM NaCl, 2 mM
KCl, 4 mM MgCl2, 1 mM EGTA, 35 mM sucrose and 5 mM HEPES pH
7.2 (Demontis and Perrimon, 2009). Buffer without EGTA was used for
measurements of contracted muscles. Body wall muscles were fixed with
4% formaldehyde in PBS for 15 min and then rinsed three times for 5 min
each in PBS with 0.5% Tween 20 (PBT). Muscles were blocked for
30 min in 20% horse serum in PBT at room temperature. Dissection and
staining of adult flies were performed as described previously (TaghliLamallem et al., 2008). Primary antibodies were applied overnight at 4°C.
After three washes in PBT, secondary antibodies were applied for 2 h at
room temperature with or without phalloidin-TRITC as appropriate.
MitoTracker Red CMXRos (Invitrogen) was used at 1.5 µM in ice-cold
Ca2+-free saline buffer; it was incubated for 15 min at 37°C with dissected
larvae. Subsequently, larvae were fixed in 4% formaldehyde for 15 min
and washed in PBS three times for 10 min each. Muscle and heart
preparations were mounted in Fluoromount-G anti-fade reagent (Southern
Biotech) and analyzed using SP5 or SP8 (Leica) confocal microscopes.
3D models of labeled muscles were generated using Imaris software
Transmission electron microscopy (TEM)
Drosophila stocks
The following Drosophila stocks were used: w1118 strain as wild type; Mef2Gal4 [Bloomington Stock Center (BSC) BL27390]; Hand-Gal4 (kindly
provided by L. Perrin, TAGC, Marseille, France); UAS-lacZ (BSC,
BL1776); UAS-RNAi-cher [Vienna Drosophila RNAi Center (VDRC)
107451]; UAS-RNAi-starvin (VDRC 42564); two UAS-RNAi-CryAB lines
(VDRC 40532 and 107305); cherCPTI0847 protein trap line [Kyoto
Drosophila Genomics Resource Center (DGRC)]; and UASp-cher90-GFP
(generated by S. Huelsmann, Gurdon Institute, Cambridge, UK). The
UASp-Venus-CryAB, UASp-Venus-CryAB ABDmut and UASp-VenusCryAB R120G lines were generated in the K.J. lab. The generation of
CryAB and cheerio transgenic lines is described in the supplementary
Materials and Methods.
TEM of dissected larvae and immunogold detection of CryAB in TEM
sections are described in the supplementary Materials and Methods.
CryAB interactions by co-IP and mass spectrometry
Primary antibodies: goat anti-GFP (1:500; Abcam, ab5450); rat anti-CryAB
(1:500; generated in the K.J. lab); rabbit anti-Mef2 (1:1000; gift from
H. Nguyen, Erlangen University, Germany); rat anti-Kettin (1:25; Abcam,
ab50585); mouse anti-LamC28.26 [1:1000; Developmental Studies
Hybridoma Bank (DSHB)]; rabbit anti-β3-tubulin (1:10,000; gift from
R. Renkawitz-Pohl, Marburg University, Germany); mouse anti-β-actin
(1:10; Invitrogen, AC-15); guinea pig anti-Msp300 (1:300; gift from
T. Volk, Weitzmann Institute, Israel); rat anti-Cheerio (1:200; Sokol and
Cooley, 1999); and rabbit anti-active Caspase 3 (1:1000; Abcam, ab13847).
We also used different secondary fluorescent antibodies as well as 18 nm
colloidal gold-AffiniPure goat anti-rat IgG (whole molecule) (Jackson
ImmunoResearch; 1:20) and 10 nm goat anti-rat IgG (whole molecule)-gold
(Sigma-Aldrich; 1:100) conjugated to Alexa Fluor 488, CY3 or CY5
(1:300; Jackson ImmunoResearch). Phalloidin-TRITC (1:1000; Sigma) was
used for revealing sarcomeric actin.
For the co-IP assay, 300 dissected and frozen Mef2>cher90-GFP (for
CryAB-Cheerio) or w1118 (for CryAB-Msp300) larvae were homogenized
at 4°C in 500 µl HEB extraction buffer [containing 50 mM Tris ( pH 7.6),
140 mM NaCl, 5 mM EDTA, 1% (V/V) NP40 and 0.5% (W/V) sodium
deoxycholate] as described previously (Perng et al., 2006) and
supplemented with a protease inhibitor cocktail (Roche). After 30 min
incubation on ice, lysate was centrifuged at 17,000 g for 10 min at 4°C.
Supernatant was pre-absorbed 1 h at 4°C with 50 µl protein G-Sepharose
(Amersham) to eliminate non-specific binding of the proteins to the beads.
The protein G-Sepharose beads were separated from the lysate by
centrifugation 1 min at 13,000 g at 4°C. The supernatant was incubated
4 h at 4°C with 5 µl rat anti-CryAB antibody or with 5 µl rat non-immune
serum as a negative control. The protein G-Sepharose was subsequently
added to each lysate/antibody mixture and incubated overnight at 4°C with
gentle agitation. The complexes were then washed three times with washing
buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.1% NP40) and proteins
eluted by boiling the beads in 2× SDS-PAGE buffer for 10 min. Eluted
proteins were separated on a 4-10% SDS-PAGE gradient gel and blotted to
PDF membrane (Whatman), blocked with 5% non-fat milk. The membrane
was incubated with anti-GFP antibody (1:5000; for CryAB-Cheerio) or with
anti-Msp300 (1:1000; for CryAB-Msp300) followed by anti-mouse or
anti-guinea pig HRP-conjugated secondary antibody (1:10,000) at 25°C for
1 h, and subsequently washed three times with TBST (TBS containing 0.5%
Tween-20). Protein bands were visualized using the ECL detection kit
(Amersham) and analyzed with the Chemi Doc imaging system (Bio-Rad).
Mass spectrometry to identify CryAB-interacting proteins is described in
the supplementary Materials and Methods.
RT-PCR analysis of CryAB gene attenuation
Analyses of CryAB transcript levels after RNAi-based attenuation are
described in the supplementary Materials and Methods.
Bioinformatics analysis of phylogenic relationships among sHsps is
described in the supplementary Materials and Methods.
Phenotypic analyses
Antibodies and fluorescent markers
Tests of larval viability and muscle performance using the righting,
motility and journey tests, and characterization of muscle morphology
and heart physiology are described in the supplementary Materials and
We thank H. Nguyen, T. Volk, R. Renkawitz-Pohl, B. Suter, L. Cooley, the BSC,
VDRC and DGRC for providing reagents and fly stocks; Fly-Facility for generation of
CryAB transgenic stocks; and J. Overton for injection of the cheerio construct.
Competing interests
The authors declare no competing or financial interests.
Author contributions
I.W. and T.J. designed the study; I.W., J.J., M.Z., O.T.-L., Y.R., M.D. and G.J.
performed the research and analyzed the data; Y.R. performed bioinformatics
analyses; I.W. and M.D. analyzed EM muscle preparations; S.H. provided cheerio
alleles; I.W., S.H., K.J. and T.J. wrote the paper.
This work was supported by Agence Nationale de la Recherche (ANR) grants MYOID and ID-CELL-SPE, an Infrastructure TEFOR grant, an Equipe Fondation pour la
Recherche Mé dicale (FRM) grant and Association Française Contre les Myopathies
(AFM) grants to K.J.
Supplementary material
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